The present invention relates to novel truncated forms of intercellular adhesion molecule (ICAM), designated "tICAMs", which effectively bind to human rhinovirus (HRV) and to lymphocyte-function associated antigen-1 (LFA-1). The present invention also pertains to DNA sequences coding for various tICAMs and to methods for preventing or amelio-rating infection and inflammation using said tICAMs.
Human rhinoviruses, the major causative agent of the common cold, belong to the picornavirus family. There are more than 100 distinct serotypes of human rhinovirus. The three-dimensional structure of several rhinovirus sero-types have now been determined to atomic resolution by Rossmann, M. G., E. Arnold, J. W. Erickson, E. W. Frankenberger, P. J. Griffith, H. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, "Structure of a common cold virus and functional relationship to other picornaviruses", Nature (1985) 317:145-153; and Kim, S., T. J. Smith, M. M. Chapman, M. G. Rossmann, D. C. Pevearj, F. J. Dutko, P. J. Felock, G. D. Diana, and M. A. McKinlay, "Crystal structure of human rhinovirus serotype 1A (HRV1A)", J. Mol. Biol. (1990) 210:91-111. The virion is composed of a protein capsid of 60 protomeric units, consisting of the four protein subunits VP1-4, surrounding an RNA genome. Each of the 60 protomeric units possesses a recessed 30-angstrom-wide depression or "canyon" which encircles the five-fold axis of symmetry of each icosahedral face of the virus and which is believed to contain the site that binds to the receptor on the target cell surface [reviewed in Rossmann,, M. G., J. Biol. Chem. (1989) 264:14587-14590].
In order to infect host cells, viruses must bind to and then enter cells to initiate an infection. Since 1959, evidence has accumulated in the literature indicating that the presence of specific binding sites (receptors) on host cells could be a major determinant of tissue tropism of certain viruses. [Holland, J. J., and L. C. McLaren, "The mammalian cell-virus relationship. II. Absorption, reception, and eclipse of poliovirus by HeLa cells," J. Exp. Med. 109:487-504 (1959); Holland, J. J., "Receptor affinities as major determinants of enterovirus tissue tropisms in humans," Virology 15:312-326 (1961)]. Specific binding to host cells has been demonstrated among picornaviruses such as poliovirus, Coxsackie virus, and rhinoviruses. By competition experiments, it has been demonstrated that some of these receptors are distinct from one another in that the saturation of the receptor of one virus had no effect on the binding of a second virus. [Lonberg-Holm, K., R. L. Crowell, and L. Philipson. "Unrelated animal viruses share receptors," Nature 259:679-681 (1976)].
Rhinoviruses can be classified according to the host cell receptor to which they bind. Tomassini, J. E. and R. J. Colonno, "Isolation of a receptor protein involved in attachment of human rhinoviruses," J. Virol. 58:290 (1986) reported the isolation of a receptor protein involved in the cell attachment of HRV. Approximately 90% of the more than 115 serotypes of rhinoviruses, as well as several types of Coxsackie A virus, bind to a single common receptor termed the "major" human rhinovirus receptor (HRR) [Abraham, G., and R. J. Colonno, "Many rhinovirus serotypes share the same cellular receptor," J. Virol. 51:340-345 (1984)]; the remaining 10% bind to one or more other cell receptors.
The major human rhinovirus receptor has been transfected, identified, purified, and reconstituted as described in co-pending U.S. patent applications Ser. No. 07/262,428 and 07/262,570, both filed Oct. 25, 1988. Greve, J. M., G. Davis, A. M. Meyer, C. P. Forte, S. C. Yost, C. W. Marlor, M. E. Kamarck, and A. McClelland, "The major human rhinovirus receptor is ICAM-1," Cell 56:839-847 (1989), identified the major HRR as a glycoprotein with an apparent molecular mass of 95 kD and having an amino acid sequence essentially identical to that deduced from the nucleotide sequence of a previously described cell surface protein named intercellular adhesion molecule (ICAM-1). ICAM-1 had first been identified based on its role in adhesion of leukocytes to endothelial cells [Rothlein, R., et al., J. Immunol. 137:1270-1274 (1986); see also Simmons, D., M. W. Makgoba, and B. Seed, "ICAM-1, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM", Nature (1988) 331:624-627; Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, and T. A. Springer, "Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families," Cell (1988) 52:925-933.] Induction of ICAM-1 expression by cytokines during the inflammatory response may regulate leukocyte localization to inflammatory sites. Subsequently, Staunton, D. E., et al., Cell 56:849 (1989) confirmed that ICAM-1 is the major cell surface receptor for HRV. See also Staunton, D. E., M. L. Dustin, H. P. Erickson, and T. A. Springer, "The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus," Cell (1990) 61:243-254.
European Patent Application 0 289 949 describes membrane-associated ICAM-1, which mediates attachment of many cell types, including endothelial cells, to leukocytes expressing lymphocyte function associated molecule-1 (LFA-1; CD18/CD11a, a member of the beta-2 integrin family). Said patent application provides a discussion of the prior research in the field of intercellular adhesion molecules.
Heterotypic binding of the leukocyte integrin LFA-1 to ICAM-1 mediates cellular adhesion of diverse cell types and is important in a broad range of immune interactions [Marlin, et al., Cell (1987) 51:813-819]. ICAM-1 also binds to MAC-1 (CD18/CD11b), another beta-2 integrin, but not to p150/95 (CD18/CD11c) [Staunton et al., Cell (1988) 52:925-933]. MAC-1 and p150/95 differ from LFA-1 by their alpha subunit. Although minimal peptide recognition sites have been identified for many other integrins, the recognition site for LFA-1 on ICAM-1 remains obscure. Staunton, et al., Cell (1990) 61:243-254 have reported that a transmembrane form of the first two domains of ICAM-1 retains some LFA-1-binding activity and that a number of mutations in the first two domains of the full-length molecule cause reductions in LFA-1-binding activity.
The primary structure of ICAM-1 is homologous to two other cellular adhesion molecules: neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG). This suggests that ICAM-1 is a member of the immunoglobulin supergene family [Simmons, et al., Nature (1988) 331:624-627; Staunton et al., Cell (1988) 52:925-933]. The CDNA sequences for ICAM-1 are described in the above-referenced papers by Simmons et al. and Staunton et al., from which the amino acid sequence of ICAM-1 has been deduced.
ICAM-1 is an integral membrane protein 505 amino acids long [SEQ ID NO: 1; encoded by nucleotides 139-1653 of SEQ ID NO: 2] and has: i) five immunoglobulin-like extra-cellular domains at the amino-terminal (extracellular) end (designated domain I [amino acid residues 1-88], domain II [89-185], domain III [186-283], domain IV [284-385], and domain V [386-453]); [Staunton, et al., Cell (1988) 52:925-933]; ii) a hydrophobic transmembrane domain (454-477); and iii) a short cytoplasmic domain at the carboxy-terminal end (478-505). The sequences of the first three domains can be aligned with immunoglobulins in a manner consistent with structural homology to the IgG constant region fold [reviewed by Williams, A. F. and A. N. Barclay, "The immunoglobulin superfamily--domains for cell surface recognition", Ann. Rev. Immunol. (1988) 6:381-405]. The IgG fold consists of two beta sheets comprised of anti-parallel beta strands A. B, E and D on one face and C, F, and G on the other (see FIG. 1). The sheets interact to form a hydrophobic interior and individual strands are linked by loops of variable length. The N-terminal loops of antibody variable regions form the antigen combining site. As shown in Example 7. below, circular dichroism spectra of the extracellular portion of ICAM-1 indicate that the molecule contains substantial amounts of beta structure, supporting the proposed IgG-fold structure for ICAM-1 domains.
As mentioned above, the three-dimensional structure of HRV-14 which binds to ICAM-1 and of ERV-1A which binds to the as yet unidentified minor receptor have been determined [Rossman, M. G., E. Arnold, T. W. Erickson, E. W. Frankenberger, P. J. Griffith, H. Hecht, J. E. Johnson, G. Kamer, M. Luc, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, "Structure of a common cold virus and functional relationship to other picornaviruses", Nature (1985) 317:145-153; Kim, S., T. J. Smith, M. M. Chapman, M. G. Rossmann, D. C. Pevear, F. J. Dutko, P. J. Felock, G. D. Diana, and M. A. McKinlay, "Crystal structure of human rhinovirus serotype 1A (HRV1A)", J. Mol. Biol. (1989) 210:91-111] and a "canyon" model of the viral binding site has been proposed. Residues in the lower part of the canyon are inaccessible to antibody molecules and thus the conserved receptor binding determinants could escape immune surveillance. Support for the canyon hypothesis comes from site-directed mutagenesis of canyon residues which alter the receptor binding properties of HRV-14 [Colonno, R. J., J. H. Condra, S. Mizutani, P. L. Callahan, M. E. Davies, and M. A. Murcko, "Evidence for the direct involvement of the rhinovirus canyon in receptor binding", Proc. Natl. Acad. Sci. USA (1988) 85:5449-5453], and from studies with capsid-binding drugs which induce a conformational change in the floor of the canyon and prevent receptor binding [Pevear, D. C., M. J. Fancher, P. J. Felock, M. G. Rossmann, M. S. Miller, G. Diana, A. M. Treasurywala, M. McKinlay, and F. J. Dutkor "Conformational change in the floor of the human rhinovirus canyon blocks adsorption to HeLa cell receptors," J. Virol. (1989) 63:2002-2007]. The dimensions of the rhinovirus canyon are sufficiently large to accommodate a single unpaired IgG domain, and it has recently been shown by electron microscopy that ICAM-1 and the related adhesion molecule NCAM have long elongated structures consistent with an end-to-end arrangement of-unpaired IgG domains [Staunton et al., Cell (1990) 61:243-254; Becker, J. W., H. P. Erickson, S. Hoffman, B. A. Cunningham, and G. M. Edelman, "Topology of cell adhesion molecules", Proc. Natl. Acad. Sci. USA (1989) 86:1088-1092]. The N-terminal domain of ICAM-1 is therefore likely to project furthest from the cell surface and be most accessible to virus. Furthermore, ICAM-1 is heavily glycosylated with the exception of the first domain. While the precise configuration of the virus-binding site on ICAM-1 remains to be determined, Staunton et al., Cell (1990) 61:243-254 have shown by site-directed mutagenesis and construction of a human/mouse chimera that the rhinovirus binding site is contained within the first two N-terminal domains of ICAM-1. A three-dimensional model of the first domain of ICAM-1 based on alignment with known immunoglobulin structures was docked with the rhinovirus canyon and used to predict possible contact residues [Giranda, V. L., M. S. Chapman, and M. G. Rossmann, "modeling of the human intercellular adhesion molecule-1, the human rhinovirus major group receptor", Proteins (1990) 7:227-233].
Several approaches to decreasing infectivity of viruses in general, and of HRV in particular, have been pursued including: i) developing antibody to the cell surface receptor for use in blocking viral binding to the cell; ii) using interferon to promote an anti-viral state in host cells; iii) developing various agents to inhibit viral replication; iv) developing antibodies to viral capsid proteins/peptides; and v) blocking viral infection with isolated cell surface receptor protein that specifically blocks the binding domain of the virus.
In 1985, the isolation of a monoclonal antibody that appeared to be directed against the major rhinovirus receptor was described. [Colonno, R. J., P. L. Callahan, and W. J. Long, "Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses," J. Virol. 57:7-12 (1986)]. This monoclonal inhibited infection of cells with the appropriate serotypes of rhinovirus and it inhibited binding of radiolabeled rhinovirus to cells. Colonno et al. subsequently reported that the monoclonal antibody bound to a protein with an apparent molecular weight of 90 kD [Tomassini, et al., J. Virol. (1986) 58:290-295]. This monoclonal antibody has been utilized in clinical trials with primates and humans and is understood to provide some protection against rhinovirus infection.
There are several other reports of attempts at therapeutic intervention in rhinovirus infections. Intranasal application of interferon in humans has been attempted. [Douglas, R. M. et al., "Prophylactic efficacy of intranasal alpha2-interferon against rhinovirus infections in the family setting," N. Eng. J. Med. 314:65-75 (1986)]. In this case, significant reduction in the severity of the infection was found, although nosebleeds were observed as a side-effect. Also, several analogs of disoxaril ("WIN" compounds) that reduce the infectivity of a number of picornaviruses (with widely varying effectiveness, depending on the serotype) have been tested in tissue culture and in some animal models [Fox, M. P., M. J. Otto, and M. A. McKinlay, Antimicrob. Ag. and Chemotherapy (1986) 30:110-116]. These compounds appear to inhibit replication at a step subsequent to receptor binding, probably at some step of virus uncoating. The atomic coordinates of the binding sites of these compounds within the viral capsid of the serotype HRV14 have been determined by x-ray crystallography, and are located in a hydrophobic pocket present in each protomeric unit of the capsid [Smith, T. J., et al., "The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating," Science (1986) 233:1286-1293]. The specific function of the binding pocket, if any, is unknown, but drug-resistant mutants with a single amino acid interchange in this region arise at high frequency and are viable [Badger, J., et al., "Structural analysis of a series of antiviral agents complexed with human rhinovirus 14," Pas 85:3304-3308 (1988); see also Dearden et al., Arch. Virol. (1989) 109:71]. This result calls into question the efficacy of such compounds as drugs.
The production of anti-peptide antibodies in rabbits has been reported using peptides derived from amino acid sequences of the viral capsid proteins that line the "receptor canyon" of HRV14 [McCray, J., and G. Werner, "Different rhinovirus serotypes neutralized by antipeptide antibodies," Nature (1987) 329:736-738]. While the titers of these sera are quite low, cross-serotype protection of cells in tissue culture from rhinovirus infection was demonstrated, raising the possibility of a vaccine.
It is an object of the present invention to provide an HRV receptor protein having the property of blocking HRV infection. Given the high affinity the virus has for its receptor, a therapeutic agent effective against HRV infection is the receptor itself, or more specifically, the virus-binding domain of the receptor. A protein, protein fragment, or peptide that comprises the virus-binding domain can block the ability of virus to bind to host cells by occupying (blocking) the receptor-binding cleft on the virus. Furthermore, since such a molecule makes some or all of the molecular contacts with the virus capsid that the receptor does, any viral mutations that would evade binding of the therapeutic molecule would also adversely affect binding of the natural receptor, and thus would be are deleterious or lethal for the virus; therefore, the likelihood of drug-resistant mutants is very low.
Using this approach, Greve, et al., Cell (1989) 56:879, [U.S. Ser. No. 07/239,571, filed Sep. 1, 1988] showed that purified transmembrane ICAM-1 (tmICAM-1) could bind to rhinovirus HRV3 in vitro. Other results with HRV2, HRV3, and HRV14 demonstrated a positive correlation between the ability to bind to rhinovirus and the ability to neutralize rhinovirus. Results using HRV14 and HRV2 demonstrated a positive correlation between the receptor class of the virus and the ability to bind to tmICAM-1 in vitro. That is, ICAM-1, being the major receptor, binds to HRV3, HRV14, and other major receptor serotypes and neutralizes them, while it does not bind or neutralize HRV2, a minor receptor serotype. Further studies, using purified tmICAM-1, demonstrated that it effectively inhibits rhinovirus infectivity in a plaque-reduction assay when the rhinovirus is pretreated with tmICAM-1 (50% reduction of titer at 10 nM receptor and one log reduction of titer at 100 nM receptor protein). These data were consistent with the affinity of rhinovirus for ICAM-1 of HeLa cells, which has an apparent dissociation constant of 10 nM, and indicates a direct relationship between the ability of the receptor to bind to the virus and to neutralize the virus.
The ICAM of the prior art is an insoluble molecule which is solubilized from cell membranes by lysing the cells in a non-ionic detergent. Because large-scale production of tmICAM-1 is not presently economically feasible, and because maintenance of tmICAM-1 in an active form requires the use of detergents, alternate soluble forms of receptor protein suitable for use as a rhinovirus inhibitor are desirable. See generally copending applications U.S. Ser. No. 07/130,378; U.S. Ser. No. 07/239,571; U.S. Ser. No. 07/262,428; U.S. Ser. No. 262,570; U.S. Ser. No. 07/301,192; U.S. Ser. No. 07/390,662; U.S. Ser. No. 07/449,356; U.S. Ser. No. 07/678,909; and U.S. Ser. No. 07/631,313, all incorporated herein by reference.
U.S. Ser. No. 07/130,378 (filed Dec. 8, 1987) and its CIP application U.S. Ser. No. 07/262,570 are directed to transfected non-human cell lines which express the major human rhinovirus receptor (HRR), and to the identification of HRR as intercellular adhesion molecule (ICAM).
U.S. Ser. No. 07/301,192 (filed Jan. 24, 1989) and its CIP applications U.S. Ser. No. 07/445,951 (abandoned) and U.S. Ser. No. 07/449,356 are directed to a naturally-occurring soluble ICAM (sICAM) related to but distinct from tmICAM in that said sICAM lacks the amino acids spanning the hydrophobic transmembrane region and the carboxy-terminal cytoplasmic region; in addition this sICAM has a novel sequence of 11 amino acids at its C-terminus.
Parent application U.S. Ser. No. 07/239,571 (filed Sep. 1, 1988) and its CIP applications U.S. Ser. No. 07/262,428 and U.S. Ser. No. 07/390,662 (abandoned in favor of continuation U.S. Ser. No. 07/678,909), and U.S. Ser. No. 07/631,313 are directed to the use of detergent-complexed tmICAM as an inhibitor of HRV infectivity, using the non-ionic detergent complex to maintain the transmembrane protein in solution. These cases are also directed to truncated forms of ICAM (tICAMs) comprising one or more of the extracellular domains I, II, III, IV, and V of tmICAM, which truncated forms do not require the presence of non-ionic detergent for solubilization. See FIG. 2.
Parent application U.S. Ser. No. 07/556,238 (filed Jul. 20, 1990) is directed to multimeric configurations of tmICAM-1 and tICAM-1 having improved ability to prevent HRV Infection. When tmICAM-1 or tICAMs are presented in a dimer configuration, the virus-binding activity of the dimerized ICAM becomes comparable to that of native tmICAM-1. This binding of multimeric ICAMs to HRV has the same properties as the binding of HRV to ICAM-1 on HeLa cells: it is inhibited by anti-ICAM-1 monoclonal antibodies, it is specific for HRV of the major receptor group, and it has the same temperature-dependence pattern as the binding of HRV to cells (i.e., binds well at 37.degree. C. and undetectably at 4.degree. C.).
The present application contains further data demonstrating the properties and efficacy of various forms of tICAM.